Continuing support is proposed for the Laboratory for Fluorescence Dynamics to expand technological biomedical research to new fields of discovery and applications and to strengthen collaborative, service, training and dissemination activities for the benefit of the biomedical research community. Fluorescence imaging and fluorescence methodologies have reached a prominent role in Biology, Biophysics and in Clinical Research. The LFD is a biotechnological resource center with a history of successes in developing new fluorescence technologies for biology and medicine and with a strong commitment to service, training, and dissemination components aimed at bringing fluorescence methods in a user facility available to the biomedical research community. For this proposal we have conceived a strong technical development program to expand current limits of imaging fluorescence instrumentation, particularly for the study of dynamical cellular processes. Projects include: developments of new microscope platforms for imaging in deep tissues, still maintaining diffraction limited resolution; exploiting the light sheet microscope for fluctuation analysis with the potential for developing radical new approaches to measure molecular flow in cells and tissue at the nanoscale; using new electronics and optics for 3D imaging of cellular dynamics; expanding the phasor approach to fluorescence lifetime imaging analysis to spectral and diffusion analysis for fast, fit-free visualization of dynamical processes and expanding our software to allow new algorithms for data acquisition and analysis in our new microscopes. The organization structure of the LFD allows users and collaborators to access innovative technologies only available in this BTRR resource, which affords them a unique opportunity to rapidly advance their own research programs. A rich program of driving biological problems exploit and push technological advances and research in biological processes, macromolecular assembly, tissue organization and dynamics, membrane morphology/function relationships, and innovative biomedical instrumentation for detection of bacteria in blood which could have an impact in the clinical area. Educational programs proposed for this application include hands-on training of students, postdoctoral fellows and visiting scientists, workshops and specialized schools in innovative fluorescence methodologies. Our improved web site will continue to be a reference source for information about fluorescence dynamic techniques, and a large repository of publications, lectures and tutorials.